Article DOI: http://dx.doi.org/10.3201/eid2112.150600 Tembusu-Related in Ducks, Thailand

Technical Appendix

Methods

Outbreak Investigations During August 2013–September 2014, we investigated outbreaks of a contagious duck disease among ducks characterized by severe neurologic dysfunction and dramatic decreases in egg production in layer and broiler duck farms in Thailand. Epidemiologic information, clinical observations, postmortem examinations, samples collection, and laboratory testing were recorded and analyzed to determine the etiology of the outbreaks.

Virus Isolation and Identification Visceral organ samples were collected from affected ducks, including brain, spinal cord, spleen, lung, kidney, proventiculus, and intestine. Each sample was homogenized in sterile phosphate-buffered saline at a 10% suspension (w/v), centrifuged at 3,000 × g for 15 min, then filtered through 0.2-μm filters. The filtered suspensions were inoculated into the allantoic cavities of 9-day-old embryonated chicken eggs. The allantoic fluids and tissue suspensions were then examined for the presence of duck Tembusu (DTMUV) by reverse transcription PCR (RT-PCR) by using E gene–specific primers (1).

The samples were also tested for avian influenza virus (2), Newcastle disease virus (3,4), and duck herpesvirus (5) to rule out other common that can cause similar symptoms. The tissue suspensions and virus isolates were also tested by hemagglutination tests against 1% chicken erythrocytes at 25°C, pH 7.4 to exclude avian hemagglutinating viruses, including avian influenza virus and Newcastle disease virus.

Whole-Genome Sequencing and Phylogenetic Analysis of Thai DTMUV In this study, 1 DTMUV isolate from Thailand (DK/TH/CU-1) was selected and subjected to whole-genome sequencing. Additionally, 5 other isolates (DK/TH/CU-2,

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DK/TH/CU-3, DK/TH/CU-4, DK/TH/CU-5, and DK/TH/CU-6) were subjected to partial E gene sequencing. Viral RNAs were extracted from allantoic fluid by using NucleoSpin Extract Viral RNA Kit (Macherey-Nagel, Düren, Germany) in accordance with the manufacturer’s instructions. RT-PCR was performed in a single-step reaction by using the AccessQuick RT- PCR System (Promega, Madison, WI, USA). Primers were designed on the basis of the complete genome sequences of DTMUV available in GenBank. The oligonucleotide primer sequences are shown in online Technical Appendix Table 1. The amplicons were then subjected to DNA sequencing (1st Base Laboratories Sdn Bhd, Malaysia). The validated nucleotide sequences were assembled by using SeqMan software v.5.03 (DNASTAR Inc., Madison, Wisconsin, USA). These nucleotide sequences of the Thai DMTUV were submitted to GenBank under accession nos. KR061333–8.

Phylogenetic analysis was performed by comparing the nucleotide sequences of polyprotein, E gene and NS genes of the Thai DMTUVs with those of other reference available in GenBank. The nucleotide sequences were aligned by using Muscle v.3.6 (7). The phylogenetic trees were constructed in MEGA v.6.0 by using the neighbor-joining and maximum-likelihood algorithms (8). To determine nucleotide identities, the nucleotide sequences of Thai, Malaysian, and Chinese DTMUVs were aligned and compared in MegAlign software v.5.03 (DNASTAR Inc.) with other reference flaviviruses.

References

1. Su J, Li S, Hu X, Yu X, Wang Y, Liu P, et al. Duck egg-drop syndrome caused by BYD virus, a new Tembusu-related flavivirus. PLoS ONE. 2011;6:e18106. PubMed http://dx.doi.org/10.1371/journal.pone.0018106

2. Spackman E, Senne DA, Myers TJ, Bulaga LL, Garber LP, Perdue ML, et al. Development of a real- time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol. 2002;40:3256–60. PubMed http://dx.doi.org/10.1128/JCM.40.9.3256-3260.2002

3. Ke GM, Yu SW, Ho CH, Chu PY, Ke LY, Lin KH, et al. Characterization of newly emerging Newcastle disease viruses isolated during 2002–2008 in Taiwan. Virus Res. 2010;147:247–57. PubMed http://dx.doi.org/10.1016/j.virusres.2009.11.008

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4. Liu H, Wang Z, Wu Y, Zheng D, Sun C, Bi D, et al. Molecular epidemiological analysis of Newcastle disease virus isolated in China in 2005. J Virol Methods. 2007;140:206–11. PubMed http://dx.doi.org/10.1016/j.jviromet.2006.10.012

5. Li H, Liu S, Kong X. Characterization of the genes encoding UL24, TK and gH proteins from duck enteritis virus (DEV): a proof for the classification of DEV. Virus Genes. 2006;33:221–7. PubMed http://dx.doi.org/10.1007/s11262-005-0060-6

9. Huang X, Han K, Zhao D, Liu Y, Zhang J, Niu H, et al. Identification and molecular characterization of a novel flavivirus isolated from geese in China. Res Vet Sci. 2013;94:774–80. PubMed http://dx.doi.org/10.1016/j.rvsc.2012.11.014

7. Edgar RC. MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 2004;5:113. PubMed http://dx.doi.org/10.1186/1471-2105-5-113

8. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9. PubMed http://dx.doi.org/10.1093/molbev/msr121

Technical Appendix Table 1. Primers used for whole-genome sequencing of a Thai duck Tembusu virus Primer name Sequence (5′3′) Reference P1f AGAAGTTCRYCTGTGTGA (6) DF_R638 CAGCAGTCTATGTCTTCAGG This study DF_F441 CGATAGTTGCTGGGCTGAAGC This study DF_R1115 GCAGTAAGATCTCACAACCGC This study DF_F954 GCTTCAGCTGTCTGGGGATGC This study DF_R1650 CAATGACTCTTTGTTTTGCCACG This study DF_F1480 CTACACYGCTGAGATGGAGG This study

DF_R2463 GCCAAGTCGATTGAGCACCCC This study DF_F2353 GGCACTGCTATTGTGGATGGG This study DF_R3339 GGTGGGGTGGTGCAAGACC This study DF_F3302 GGAACAACTGTCACAGTAACG This study DF_R4694 GCATGACTCCCACTCCAGCC This study DF_F4406 GCATCACAGAGATTTGATGTGG This study DF_R5162 CCTGAACCTGGATGTAGGTCC This study DF_F4874 GCAAGTCATCGTCGTGCAACC This study DF_R5582 GCTCTTCAATGTCTGTTATTGGC This study DF_F5399 GCTCACACCTCAGCGAGTGC This study DF_R6249 GGTCATTGTAACTTATCCCAGC This study DF_F5928 CCAGTTCCTATAACATCAGCC This study DF_R6678 GCTCCAAGACCTGTCTTCCC This study DF_F6494 CGCTCACAGAATGACAGAATCC This study DF_R7348 GGAACATCTGTAGCCACTATGC This study DF_F6807 GAACCAGAGAGACAGAGATCGC This study DF_R8158 CCCTAGCTAGCCATTCCTCGG This study DF_F7940 GCAGGTTCAGGAAGTGAGAGG This study DF_R8536 GGATTGTCTTGGTCATAATGCC This study DF_F8383 GGATGCACAAAACCAACCGC This study DF_R9215 GGCCGAGATGTCACGCAGC This study DF_F8084 GCTGTGTGACATAGGTGAAGC This study DF_R9449 CCACTTCCCCTCTGGTCTTCC This study DF_F9274 GGGACACTAGAATAACCAAGGC This study DF_R10485 CCAACATCCGGTGGCAGGG This study

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Technical Appendix Table 2. Nucleotide identities of the polyprotein gene sequences of a Thai DTMUV (DK/TH/CU-1) with reference viruses of Ntaya virus group DEDSV- DK/TH/CU-1 WFZ_2012 CJD05 xz_2012 YY5 goose pigeon byd1 BYD-1 MM1775 STWV NTAV BAGV ITV ROCV ILHV ZIKV DK/TH/CU-1 100 97.7 97.6 97.7 97.9 97.8 97.9 97.9 97.9 90.3 89.4 76.9 76.7 77 69.4 68.8 65.2 WFZ_2012 99.1 99.2 99.4 99.3 99.3 99.4 99.4 90.6 89.4 76.9 76.5 77 69.4 68.6 65.2 CJD05 99.1 99.3 99.2 99.3 99.4 99.4 90.6 89.3 76.9 76.5 76.8 69.3 68.6 65.3 xz_2012 99.4 99.3 99.3 99.4 99.4 90.7 89.5 77 76.5 76.8 69.3 68.7 65.4 YY5 99.5 99.6 99.6 99.6 90.7 89.5 77 76.6 76.9 69.4 68.7 65.3 goose 99.9 99.6 99.6 90.6 89.5 77 76.5 76.8 69.4 68.7 65.3 pigeon 99.6 99.6 90.7 89.5 77 76.5 76.9 69.4 68.7 65.3 DEDSV-byd1 100 90.7 89.5 77 76.6 76.9 69.4 68.7 65.3 BYD-1 90.7 89.5 77 76.6 76.9 69.4 68.7 65.3 MM1775 94 76.5 76.6 76.9 69.3 68.6 65.1 STWV 76.5 76.5 76.8 69.2 68.4 65.1 NTAV 79.9 80.2 69 68.5 65.2 BAGV 94.9 69.3 69.1 65.2 ITV 69.6 69.1 65.2 ROCV 74.9 65.4 ILHV 65.4 ZIKV 100 *BAGV, Bagaza virus; DK/TH/CU-1, Thai duck tembusu virus; ILHV, Ilheus virus; ITV, Israel turkey meningoencephalomyelitis virus; WFZ_2012, CJD05, xz_2012, YY5, goose, pigeon, DEDSV-byd1, BYD-1, Chinese duck tembusu viruses; MM1775, Tembusu virus strain MM1775; NTAV, Ntaya virus; ROCV, Rocio virus; STWV, Sitiawan virus; ZIKV, Zika virus.

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A

B

60.0

50.0

40.0

30.0

20.0

10.0 DTMUV prevalence (%)

0.0 Aug-13 Sep-13 Oct-13 Nov-13 Dec-13 Jan-14 Feb-14 Mar-14 Apr-14 May-14 Jun-14 Jul-14 Technical Appendix Figure 1. Details of DTMUV outbreaks in Thailand during 2013–2014. A) Locations of DTMUV outbreaks in Thailand. Circles represent provinces where the DMTUV isolated. NR, Nakhon Ratchasima; PC, Prachinburi; CB, Chonburi; SB, Suphanburi. B) Monthly prevalence of DTMUV in farmed ducks, Thailand, 2013–2014. DTMUV, duck Tembusu virus.

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TA df-2 FX 2012 BYD-1 LD 2010 86 Shandong1 XHZD/2010 Goose 100 JXSP Pigeon JS804 FS 87 JM SD/2011 Chinese DTMUV ZJ-6 TMUV JS/2010 ZJ 407 YY5 CJD05 9992 WR/2010 WFZ 2012 100 CK-SD-11 99 GS-PT-7 100 Du/CH/LSD/110128 FJMH220 99 100 100 JX2 DK/TH/CU-1 Thai DTMUV 100 MM 1775 100 Sitiawan virus Ntaya virus Bagaza virus 100 100 Israel turkey meningoencephalomyelitis virus 100 Rocio virus Ilheus virus St. Louis encephalitis virus 97 100 88 1 87 West Nile virus 2 100 Japanese encephalitis virus Murray Valley encephalitis virus 98 71 Usutu virus Kokobera virus Bussuquara virus 96 Kedougou virus Zika virus 4 Dengue virus 2 100 Dengue virus 1 99 73 Dengue virus 3 100 Sepik virus 100 Wesselsbron virus 100 virus Entebbe bat virus 100 Yokose virus 84 Rio Bravo virus 99 76 Montana myotis leukoencephalitis virus 100 Modoc virus Apoi virus 99 Karshi virus Powassan virus 100 Alkhurma virus 98 Langat virus 98 Omsk hemorrhagic fever virus 98 Louping ill virus 100 100 Tick-borne encephalitis virus 100 Culex flavivirus Quang Binh virus 100 Cell fusing agent virus Aedes flavivirus 100 91 Kamiti River virus Tamana bat virus

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Shandong1 64 LD 2010 Partial E gene BZ 2010 JS804 df-2 DEDSV-byd1 JS/2010 CK-SD-11 Goose JXSP Pigeon ZJ-6 TA BYD-1 lq-1 SX1 zc-1 62 SDXT SDSG SDLC 95 AHQY CJD05 XHZD/2010 62 JS SDMS

85 YY5 TMUV-SDHS ZJ 407 89 ZJ GH-2 GS-PT-7 Duan 69 xz 2012 White Kaiya duck/WR/China/2010 65 GX 2011 63 WFZ 2012 FS 98 77 JM FX 2012 muscovy/SD/China/2011 DK/TH/CU-6 DK/TH/CU-1 62 DK/TH/CU-3 Thai DTMUV 85 DK/TH/CU-2 DK/TH/CU-4 72 DK/TH/CU-5 JX2 DTMUV-AH2011 FJMH220 Du/CH/LSD/110128 D1921/1/4 Malaysian DTMUV 99 D1921/1/3 Tembusu virus strain MM 1775 99 Sitiawan virus

0.02

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NS5 gene JXSP Pigeon 87 Goose JS Duan XHZD/2010 ZJ-6 BYD-1 68 DEDSV-byd1 FX 2012 LD 2010 62 BZ 2010 67 77 Shandong1 CJD05 SDMS YY5 TMUV-SDHS muscovy/SD/China/2011 GX 2011 White Kaiya duck/WR/China/2010 JS/2010 ZJ 407 70 ZJ GH-2 99 SDLC AHQY JS804 86 lq-1 zc-1 SDSG SX1 91 SDXT WFZ 2012 78 xz 2012 FS 93 JM df-2 80 TA CK-SD-11 93 GS-PT-7 99 DTMUV-AH2011 Du/CH/LSD/110128 84 FJMH220 88 JX2 DK/TH/CU-1 Thai DTMUV D1921/1/4 Malaysian DTMUV 99 D1921/1/3 Tembusu virus strain MM 1775 99 Sitiawan virus

0.02

Technical Appendix Figure 2. Phylogenetic analysis of the nucleotide sequences of polyprotein gene (10,278 bp), partial E gene (361 bp), and partial NS gene (900 bp) of Thai DTMUVs and selected reference strains of flaviviruses. The nucleotide sequences were aligned by using Muscle v.3.6 (7). The phylogenetic trees were constructed in MEGA v.6.0 by using the maximum-likelihood algorithm with the Hasegawa-Kishino-Yano (HKY) model applied to 500 replications of bootstrap (8). Circle indicates Thai DTMUVs.

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